CN102772798A - Methods and compositions for increasing the efficiency of therapeutic antibodies using nk cell potentiating compounds - Google Patents

Abstract

The present invention relates, generally, to methods and compositions for increasing the efficiency of therapeutic antibodies. Their efficiency is enhanced through the increase of the ADCC mechanism. More particularly, the invention relates to the use of a therapeutic antibody in combination with compounds that block an inhibitory receptor or stimulate an activating receptor of an NK cell in order to enhance the efficiency of the treatment with therapeutic antibodies in human subjects.

Description

Use the NK cell potentiating compounds to improve the method and composition of efficiency of therapeutic antibodies

Technical field

Present invention relates in general to improve the method and composition of efficiency of therapeutic antibodies.More specifically; The present invention relates to therapeutic antibodies and the activated receptor of inhibition receptor or the stimulating natural killer cell of blocking-up natural killer cell chemical compound unite use; Thereby make the cytotoxicity effectiveness of natural killer cell of mammalian subject strengthen, especially through promoting ADCC mechanism to strengthen the therapeutic efficiency in human patients.

Background technology

Human many therapeutic schemes all are to be based upon on the basis of using therapeutic antibodies.These comprise, for example use the therapeutic antibodies of having developed to make target cell depleted, particularly the cell of diseased cells such as virus infection, tumor cell or other source of disease cell.These antibody are monoclonal antibodies of typical IgG class, are typically to have human IgG1 or IgG3 Fc part.These antibody can be natural antibody or recombinant antibodies; And normally the mouse antibodies of " humanization " (promptly; Comprise functional domain, be typically the Fc part of people or non-human primates and have the variable region or the complementary determining region (CDR) in mice source) from various species.Optionally alternately be, this monoclonal antibody can be in genetically modified mice with people Ig site through immunization full-length human, or through obtaining by cDNA library derived from human cell.

A special example of this therapeutic antibodies is Rituximab (rituximab) (Mabthera

), it be a kind of by people γ 1 and K constant region (thereby having human IgG1 Fc part) with have the chimeric anti-CD 20 monoclonal antibody that the specific Mus of CD20 variable region connects into.In in the past several years, Rituximab has greatly changed the therapeutic scheme to bone-marrow-derived lymphocyte hypertrophy property canceration, especially non-Hodgkin lymphoma (NHL).Other example of humanized IgG1 antibody comprises and is used to treat the B cell malignancies; Ah the coming of (canceration) organizes monoclonal antibody; (alemtuzumab) and be used to treat the Herceptin of breast carcinoma; (trastuzumab) other example of the therapeutic antibodies in

exploitation occurs just in the art.

The mechanism of action of therapeutic antibodies remains the topic of an arguement.Inject antibody, cause having the depletion of being discerned antigenic cell by this antibody specificity.This depletion effect can mediate through at least 3 kinds of mechanism: antibody-mediated cytotoxicity (ADCC), complement dependent form dissolving and suppress through the signal that provides as the antibody target target antigen direct antitumor to tumor growth.

Although these antibody have been represented a kind of human treatment, especially for the treatment tumor, brand-new and effective method, they also not all have very strong curative effect.For example, demonstrate for low effective with height NHL in the treatment although Rituximab is used (giving) separately or use (giving) with chemotherapy drugs in combination, 30% to 50% low NHL patient does not have clinical response for Rituximab.The low concentration of Rituximab can be interpreted as any Rituximab in some patients and is of no curative effect in high tumor load that someone proposes to exist during the CD20 expression, treatment on the lymphocytic cell surface or the serum.Yet it is unknown that the actual cause of treatment failure remains to a great extent.

In addition, because the side effect of administering therapeutic property antibody makes the use of therapeutic antibodies be restricted.For example, the heating that in the patient, occurs, headache, feel sick, possible dosage or frequency that hypotension, asthma, erythra, infection and many side effect that other possibly occur have limited administration of antibodies potentially.

Therefore, realizing that curative effect reduces issuable side effect as far as possible thereby the curative effect of raising therapeutic antibodies perhaps can reduce the using dosage of antibody, is interesting.The present invention will address these problems and other needs.

Summary of the invention

The present invention has disclosed the new method that strengthens efficiency of therapeutic antibodies.Need not receive the restriction of following theory, we think use the resultant surprising result of the inventive method to be because: when injecting therapeutic antibodies, they can strengthen ADCC mechanism in vivo.In fact, brand-new compositions provided by the invention can overcome the difficulty relevant with the therapeutic antibodies curative effect at present with method.Can see from the NK cell of individuality owing to lack the activation of NK cell, and having the ADCC of very poor therapeutic monoclonal antibodies (mAb) mediation in the present invention promptly through at the inhibition receptor that suppresses on the NK cell.Preferably; The raising of ADCC mechanism is through using blocking-up at the inhibition receptor on the natural killer cell or stimulate the chemical compound of the activated receptor on natural killer cell, thereby promotes the cytotoxic effect of mammalian subject enhanced natural killer cell to realize.Preferred chemical compound is antibody or its fragment.

Said antibody or other chemical compound can (for example killer cell inhibition receptor (KIR or NKG2A/C) molecule reacts with NK cell inhibiting property receptor; Or with its activated receptor (the for example such NCR of NKp30, NKp44 or NKp46 on the NK cell) reaction, thereby in the cell inhibiting effect and to have increased its ADCC active.

More specifically, the present invention has disclosed treatment patient's method, block N K cell inhibition receptor or stimulate the chemical compound of its activated receptor wherein, and preferably antibody or its fragment are co-administered in the patient with therapeutic antibodies.Among this paper the inventor proved therapeutic antibodies through as altogether injection system be preferably antibody or its segmental this compounds is co-administered; Activated receptor through for example block N K cell inhibiting property receptor or stimulation NK cell has overcome the NK cell inhibiting, thereby can greatly strengthen its therapeutic efficiency.

The invention still further relates to the pharmaceutical composition that comprises therapeutic antibodies and block N K cell inhibiting property receptor or stimulate the chemical compound of NK cell activation receptor, this chemical compound is preferably antibody or its fragment.The invention still further relates to the test kit of the chemical compound of the activated receptor that comprises therapeutic antibodies and block N K cell inhibiting property receptor or stimulate the NK cell, this chemical compound is preferably antibody or its fragment.

The invention still further relates to block N K cell inhibiting property receptor or stimulate the chemical compound (being preferably antibody or its fragment) of the activated receptor of NK cell to be used to increase the therapeutic efficiency of therapeutic antibodies, or be used to improve the application of the intravital ADCC of patient that treats with therapeutic antibodies.

The invention still further relates to block N K cell inhibiting property receptor or stimulate the chemical compound (being preferably antibody or its fragment) of its activated receptor and the application that therapeutic antibodies is used for preparing the medicine of treating disease.More specifically, this treatment of diseases needs to eliminate (exhausting) target cell, is preferably ill cell, like cell, tumor cell or other pathogenic cell of viral infection.Preferably, this disease is cancer, infectious disease or immunological diseases.More preferably, this disease is selected from and comprises cancer, autoimmune disease, inflammation and one group of viral disease.This disease also relates to transplant rejection, and more concrete is allograft rejection and graft versus host disease (GVHD).

The present invention also comprises the method that is used to reduce therapeutic antibodies (for example through the antibody of Fc γ receptors bind, being preferably CD16 (Fc γ RIIIa)) dosage.For example, the co-administered therapeutic antibodies that can allow to use low dosage of the chemical compound of the active acceptor on inhibition receptor on therapeutic antibodies and the block N K cell or the stimulation NK cell.This antibody-like can the RD with than this chemical compound not the time lack 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or lower dosage use.

In addition, the invention provides the effective therapeutic dose and the method that reduces dosage that is used to measure therapeutic antibodies (for example by the bonded antibody of CD16).This method comprises: i) therapeutic antibodies of first concentration and target cell and NK cell are hatched jointly, and do not have the inhibition receptor on the block N K cell or stimulate the chemical compound of the activated receptor on it to exist; Ii) the therapeutic antibodies of second low concentration and target cell and NK cell are hatched jointly, and have the inhibition receptor on the block N K cell or stimulate the chemical compound of the activated receptor on it; Iii) determination step ii) in viewed target cell elimination (exhaustion) whether with step I) in viewed elimination as many.If observe step I i) and step I) effectively same; Can change the relative concentration of chemical compound and therapeutic antibodies so; And observe (exhaustion) situation of elimination; Thereby confirm to be applicable to the different condition of particular patient, for example farthest eliminate target cell, and reduce the dosage of therapeutic antibodies as far as possible or reduce the dosage of chemical compound as far as possible to patient's different needs.

A particular aspects, the invention provides a kind ofly in the method that the human patients interior therapeutic disease that needs is arranged, comprising: a) said patient is used block N K cell inhibiting property receptor or stimulate the chemical compound of its activated receptor; And b) said patient is used and can carry out bonded therapeutic antibodies through CD16.

In one embodiment, this therapeutic antibodies and chemical compound are applied to the patient simultaneously.In another embodiment, at administering therapeutic property antibody 1 in week, in 4 days, in 3 days or same in a few days (in promptly 24 hours) with this compound administration in this patient.In another embodiment, this disease is cancer, infectiousness (infectivity) disease or immunological diseases.

In one embodiment, this method further comprises an additional step, wherein intravital NK cell activity of measuring patient or quantity before or after using this chemical compound.In another embodiment, this additional step comprises: i) before dispenser, in patient's body, obtain the NK cell; Ii) under the situation that this chemical compound/this chemical compound of nothing is arranged, the NK cell is hatched under the situation that has one or more target cells that can be discerned by this therapeutic antibodies; And iii) measure this chemical compound is eliminated (consumption) target cell ability to the NK cell influence; Show that this chemical compound is applicable to this method when finding that this chemical compound has strengthened when the NK cell is eliminated the ability of target cell, and this method is applicable to this patient.

On the other hand; The invention provides a kind of pharmaceutical composition; Comprise: a therapeutic antibodies (for example can pass through the bonded antibody of CD16), a chemical compound that suppresses NK cell inhibiting property receptor or stimulate its activated receptor, and the acceptable carrier of medicine.On the other hand, the invention provides a kind of test kit, comprising: a therapeutic antibodies (for example through the bonded antibody of CD16), and one or more block N K cell inhibiting property receptor or stimulate the chemical compound of its activated receptor.

For any said method, compositions or test kit, this therapeutic antibodies has human IgG1 or IgG3 Fc fragment in one embodiment.In another embodiment, this chemical compound is antibody or its fragment.In another embodiment, this therapeutic antibodies is monoclonal antibody or its fragment.In another embodiment, this therapeutic antibodies does not partly combine with radioactive segment or toxicity.In another embodiment, this chemical compound suppresses NK cell inhibiting property receptor.In another embodiment, this chemical compound stimulates the activated receptor of NK cell.In another embodiment, this chemical compound is people's antibody, humanized antibody or chimeric antibody, or its fragment.In another embodiment, this therapeutic antibodies or chemical compound can be antibody fragment or the derivants like Fab fragment, Fab ' 2 fragments, CDR and ScFv.

In one embodiment, this therapeutic antibodies is people's antibody, humanized antibody or chimeric antibody, or its fragment.In another embodiment, this therapeutic antibodies is Rituximab (rituximab) or Kan Pasi (Campath).In another embodiment, this antibody is Rituximab, and said antibody is to be less than 375mg/m weekly ²Dosage use.In another embodiment, this antibody is Kan Pasi, and this antibody is used with the dosage that is less than 90mg weekly.

In one embodiment, at least one in this chemical compound and NKG2, KIR2DL or the KIR3DL people's receptor combines, and suppresses the cytotoxicity inhibitory action of the NK cell of relevant NKG2-, KIR2DL-or KIR3DL-mediation.In another embodiment, this compounds block is selected from the one group of NK cell inhibiting property receptor that comprises KIR2DL1, KIR2DL2/3, KIR2DL4, KIR2DL5A, KIR2DL5B, KIR3DL1, KIR3DL2, KIR3DL3, LILRB1, NKG2A, NKG2C, NKG2E and LILRB5.In another embodiment, this chemical compound combines with the common determinant of KIR2DL people's receptor, and suppresses the inhibitory action of the NK cell poison of KIR2DL mediation.In another embodiment, this chemical compound combines with the common determinant of KIR2DL1, KIR2DL2 and KIR2DL3 people's receptor, and suppresses the cytotoxicity inhibitory action of the NK cell of KIR2DL1-, KIR2DL2-and KIR2DL3-mediation.In another embodiment; This chemical compound is suppressed at the 80th (site 80) and has the HLA-C allele molecule of lysine (Lys) residue and combining of people KIR2DL1 receptor, and is suppressed at the 80th (80 site) and has the HLA-C allele molecule of agedoite (Asn) residue and combining of people KIR2DL2 and KIR2DL3 receptor.In another embodiment, this chemical compound is attached on the same antigenic determinant with the monoclonal antibody DF200 that is produced by hybridoma DF200.In another embodiment, this chemical compound is attached to NK cells of human beings on the KIR on surface receptor with the monoclonal antibody DF200 competition that is produced by hybridoma DF200.In another embodiment, this chemical compound is monoclonal antibody DF200 or its fragment that is produced by hybridoma DF200.

In one embodiment, this chemical compound be selected from a receptors bind in a group that comprises NKp30, NKp44, NKp46 and NKG2D.In another embodiment, this chemical compound is that a monoclonal antibody that is selected from a group that comprises AZ20, A76, Z25, Z231 and BAB281 is deutero-, or competition with it.

On the other hand, the invention provides a kind of selection and be used for the method with the co-administered chemical compound of therapeutic antibodies, said method comprises: a kind of NK of inhibition cell inhibiting property receptor i) is provided or stimulates the testing compound of its activated receptor; Ii) under the condition that the NK cell exists, when having/not having this testing compound, with therapeutic antibodies with being hatched by the target cell of this therapeutic antibodies specific recognition; And iii) measure the target cell ability of influence this chemical compound is eliminated to(for) the NK cell; When wherein measuring the ability of this chemical compound enhanced NK cell elimination target cell, show that then this chemical compound is applicable to this method.

In one embodiment, this chemical compound strengthens 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500% or higher with the ability that this therapeutic antibodies destroys target cell.In another embodiment, this chemical compound is selected from the one group of material that comprises antibody, antibody fragment, monoclonal antibody, monoclonal antibody fragment, humanized antibody, chimeric antibody and people's antibody.In another embodiment, this target cell is the cell of cancerous cell, viral infection or the cell that constitutes the autoimmune disease basis.In another embodiment, this therapeutic antibodies is Rituximab (rituximab) or Kan Pasi (CAMPATH).

On the other hand; The invention provides that a kind of raising is applied in patient's body can be by the method for the therapeutic efficiency of the bonded therapeutic antibodies of CD16, and said method is included in to be used before the said therapeutic antibodies, simultaneously or afterwards said patient is used block N K cell inhibiting property receptor or stimulate the chemical compound of effective therapeutic dose of its activated receptor.In one embodiment, this chemical compound has improved therapeutic efficiency through the ADCC that strengthens said patient.

Description of drawings

Fig. 1: monoclonal antibody DF200 combines with a common determinant of different people KIR2DL receptor.

Fig. 2: is 4/1 to dissolve reconstruct with effector/target ratio with anti-KIR2DL monoclonal antibody (anti-KIR2DL mAb) on C1R Cw4 target.Monoclonal antibody DF200 suppresses the inhibitory action of the cytotoxicity (reconstruct dissolving) of two the positive NK cell of KIR2DL1 on the Cw4 positive target cell of KIR2DL-mediation.

Fig. 3: mediate on the positive EBV cell line of a Cw4 through the Rituxan with the positive NK clone of a KIR2DL1, interacting through blocking-up KIR/HLA strengthens ADCC.To (Mabthera Rutixan) exists down with 10ug/mlEB6 antibody (anti-KIR2DL1) at the 5ug/ml anti-CD 20 antibodies; Rutixan acts on separately; EB6 acts on separately; Or having no antibody to exist down, (CD20 the is positive) target cell that transforms for the positive EBV of Cw4 with various effectors/target ratio (from 1 to 4) is the NK clone cell dissolving relation of tested K IR2DL1.When having anti-KIR2DL antibody (EB6), greatly strengthened ADCC.

Fig. 4: mediate on the positive EBV cell line of a Cw4 through the Kan Pasi (Campath) with the positive NK clone of a KIR2DL1, interacting through blocking-up KIR/HLA strengthens ADCC.To in the presence of Kan Pasi and 100ug/ml EB6 antibody (anti-KIR2DL1); The bank Paasche acts on separately; EB6 acts on separately; Or having no antibody to exist down, (CD20 is positive) target cell that the positive EBV of Cw4 is transformed is the NK clone cell dissolving relation of tested K IR2DL1.When having anti-KIR2DL1 antibody (EB6), greatly strengthened ADCC.

The specific embodiment

The invention provides a kind of method that is used to increase efficiency of therapeutic antibodies.The present invention has especially disclosed a kind of application of compound, and this chemical compound is preferably antibody or its fragment, but the effectiveness of its enhanced NK cell, preferably through block N K cell inhibiting property receptor or activate the effect that its activated receptor can significantly improve therapeutic antibodies.In fact, the inventor has proved that the curative effect of multiple therapeutic antibodies can go up the combined effect of antibody and greatly strengthens through directly acting on NK cell receptor (like, inhibition receptor).

Therefore, the present invention relates to a kind of method, comprising in patient's interior therapeutic disease that needs are arranged:

A) give said patient use a kind of can block N K cell inhibiting property receptor or stimulate the chemical compound of its activated receptor, be preferably an a kind of antibody or its fragment; And

B) said patient is used a kind of therapeutic antibodies.

Said therapeutic antibodies can combine through the CD16 of NK cell, and preferably the Fc zone through it combines.

Preferably, said therapeutic antibodies has human IgG1 and IgG3 Fc part, particularly monoclonal antibody or its fragment, further is preferably humanized antibody, people's antibody or chimeric antibody, or its fragment, for example Rituximab (rituximab).

Desirable is the chemical compound of block N K cell inhibiting property receptor, is preferably antibody or its fragment, can be before using (giving) therapeutic antibodies, be applied to (giving) patient simultaneously or afterwards.The application process of different antibodies depends on their bioavailability and pharmacokinetics.Preferably using this chemical compound administering therapeutic property antibody in one week, the chemical compound of this block N K cell inhibiting property receptor is preferably antibody or its fragment, is more preferably in 5 days or 2 days and uses.Preferably, this therapeutic antibodies is before the chemical compound of using block N K cell inhibiting property receptor or use simultaneously, is preferably antibody or its fragment.

On the other hand; The present invention relates to a kind of method that in patient's body of receiving treatment property Antybody therapy, improves ADCC; Said method comprises to be used before the said therapeutic antibodies, simultaneously or use the chemical compound of the raising ADCC of q.s afterwards to said patient; This compounds block NK cell inhibiting property receptor is preferably antibody or its fragment.Said therapeutic antibodies can combine through the CD16 on the NK cell, and preferably the Fc zone through it combines.Preferably, said therapeutic antibodies is human IgG1 or IgG3 fragment, particularly monoclonal antibody or its fragment, more preferably individual antibody, humanized antibody or chimeric antibody, or its fragment, for example Rituximab.

On the other hand; The present invention relates to the method for the intravital efficiency of therapeutic antibodies of a kind of patient of raising; Said method comprises to be used before the said therapeutic antibodies, simultaneously or use the chemical compound of a certain amount of block N K cell inhibiting property receptor afterwards to said patient; Be preferably antibody or its fragment, this dosage is enough to improve the effect of said therapeutic antibodies.Said therapeutic antibodies can combine through CD16, preferably combines through its Fc zone.Preferably, said therapeutic antibodies is human IgG1 or IgG3 Fc fragment, particularly monoclonal antibody or its fragment, more preferably people's antibody, humanized antibody or chimeric antibody, or its fragment, for example Rituximab.

Definition

In this article, following except as otherwise noted term has the implication that it defines.

Among this paper, " NK " cell is meant and the Ia lymphocyte subgroup of unconventional property.The NK cell can be discerned through certain characteristic and biological property: like the expression of (comprising CD16, CD56 and/or CD57) of specific surfaces antigen; The shortage of α/β or gamma/delta TCR complex on cell surface; Be connected to the ability of not expressing the antigenic cell of " from body " MHC/HLA and passing through to activate specific cytase cell killing; Kill the tumor cell of the part that is used to express the NK activated receptor or the ability of other diseased cells, and discharge be known as cytokine protein molecular to stimulate or to suppress the ability of immunne response.Utilize method well known in the art, any of these characteristic and activity may be used to discern the NK cell.

The term that uses among this paper " antibody " is meant polyclone or monoclonal antibody.Depend on the type of constant region on the heavy chain, antibody can be divided into 5 primary categories: IgA, IgD, IgE, IgG and IgM.In these classifications some can further be divided into subclass or isotype, like IgG1, IgG2, IgG3, IgG4 or the like.A kind of typical immunoglobulin (antibody) construction unit comprises a tetramer (tetramer).To constituting, every pair (polypeptide chain) has one to each tetramer by two pairs of same polypeptide chains " gently " chain (about 25kDa) and one " weight " chain (about 50-70kDa).The about 100-110 of the N-terminal of each chain definition or the variable region of amino acids more, the mainly responsible antigen recognition in this variable region.Variable light chain (V _L) and variable heavy chain (V _H) two terms are meant these light chains and heavy chain respectively.CH corresponding to different types of immunoglobulin is called " α, " " δ, " " ε, " " γ, " and " μ " respectively.The subunit of different classes of immunoglobulin (subunit) structure and three-dimensional conformation have been known.IgG and/or IgM are preferred antibodies classifications among the present invention, are preferably IgG especially, because they are to be modal antibody under physiological condition, because their preparations the most easily under laboratory condition, and owing to IgG can be discerned by Fc γ receptor-specific.Preferably, antibody of the present invention is monoclonal antibody.Particularly preferably be humanized antibody, chimeric antibody, people's antibody or other-people-suitable antibody.

In context of the present invention, term " a curative antibody or a plurality of antibody " is meant to have any antibody that can eliminate (apoptosis) patient body internal target cell function especially.Especially, the therapeutic antibodies specificity is attached on the antigen that is present in the target cell surface, main or unique the appearing on the tumor cell like tumor specific antigen.Preferably, therapeutic antibodies comprise people Fc part or can with people Fc acceptor interaction.Therapeutic antibodies can be through like ADCC or other the whole bag of tricks (means) alignment targets cell, and can be " exposing ", does not promptly have coupling part (conjuigated moieties), or with combine such as radioactive marker or these chemical compounds of toxin.

Term " specificity is attached to " is meant that antibody combines preferably to can be incorporated on the binding partners (binding partner) in the test in competitiveness; For example active NK receptor such as NKp30, NKp44 or the NKp46 when the native protein that exists on the surface with the recombinant forms of albumen or its epitope or isolating NK or relevant target cell is tested, or human Fc gamma receptor.Competitive combination is tested and is used to measure bonded other method of specificity and can further describe hereinafter, also is known in the field simultaneously.

The antibody that " is applicable to the people's " is meant and can be used safely in the people, any antibody in the Therapeutic Method for example described herein, derive antibody or antibody fragment.The antibody that is applicable to the people comprises all types of humanized antibodies, chimeric antibody or people's antibody completely, thereby or the part of any antibody at least derive from the people antibody or through the antibody after modification avoid when use natural non--bring out immunne response during people's antibody.

" immunogenic fragments " in this article refers to any polypeptide or the fragments of peptides that can excite immunne response; Contain the generation of (comprising that film joins receptor and reaches by its deutero-mutant) of said segmental any type of molecule like said fragment of (i) antibodies and/or combination; (ii) relate to the T-cell and comprise any MHC molecule and by the stimulation of the T-cell response of the bimolecular complex of the deutero-peptide of said fragment reaction, the (iii) combination of the carrier of transfection (as expressing the phage or the antibacterial of encoding mammalian immunoglobulin gene).Optional alternate; Immunogenic fragments also can refer to any structure that can excite immunne response as indicated above; As being attached to the fragments of peptides on the carrier protein through covalently bound son (covalently bound thing); In aminoacid sequence, comprise the chimeric recombinant polypeptide structure of said fragments of peptides, and especially comprise and use the cDNA cells transfected, comprise said segmental coding in the sequence of this cDNA.

For the object of the invention, " humanization " antibody is meant a such antibody, and wherein constant structural area and the varistructure district by one or more human normal immunoglobulin merges through the land as CDR and a kind of animal immune globulin.Design such humanized antibody and be in order to keep non-human antibody's binding specificity, therefrom to derive each land, but will avoid immunoreation for the non-human antibody.

" chimeric antibody " is meant a kind of antibody molecule; Wherein (a) constant region or its part is changed, substitute or exchange make antigen binding site (variable region) be different with one or change after class, effector function and/or kind, or the constant region of a diverse molecule link to each other, these molecules can be given brand-new performance for chimeric antibody such as enzyme, toxin, hormone, somatomedin, medicine etc.; Or (b) variable region or its part is changed, substitute or have different or exchange through the variable region of the antigenic specificity that changes with a kind of.In a preferred embodiment of the invention, chimeric antibody is still preserved the Fc zone of immunoglobulin, preferably be that people Fc is regional, thus can with the lip-deep people Fc of target cell acceptor interaction.

In context of the present invention, " enhancing, " " active, " or " activatory " NK cell are meant bioactive NK cell, and more special being meant has the NK cell of dissolving target cell ability.For example a kind of " activity " NK cell can kill expresses NK activated receptor-part, and does not express the cell of " self " MHC/HLA antigen (the incompatible cell of KIR).The instance of suitable target cell of killing experiments of being used for leading again has P815 cell and K562 cell; But a lot of cell types also can use and be known in the field (referring to, like Sivori et al. (1997) J.Exp.Med.186:1129-1136; Vitale et al. (1998) J.Exp.Med.187:2065-2072; Pessino et al. (1998) J.Exp.Med.188:953-960; Neri et al. (2001) Clin.Diag.Lab.Immun.8:1131-1135)." strengthen, " " active, " or " activatory " cell also can be discerned with active relevant other performance or the activity well known in the art of NK through any, like the increase of the endocellular liberation calcium content of cytokine (for example IFN-γ and TNF-α) generation.To the object of the invention, " strengthen, " " active, " or " activatory " NK cell refer in particular to intravital not through stimulating the inhibition receptor repressed NK cell or in this cell for example through stimulating activated receptor to overcome so inhibiting NK cell.

As used herein; Term " active NK receptor " is meant any molecule on the NK cell surface; When irriate; It causes and active relevant, the performance known in the field of NK or active measurable increase, the ability of the target cell of describing like the increase of the generation of cytokine (like IFN-γ and TNF-α), endocellular liberation calcium content, in this manual other place of in the killing experiments that leads again, leading or the ability of stimulation NK cell proliferation.Term " active KIR receptor " is including, but not limited to NKp30, NKp44, NKp46, NKG2D, IL-12R, IL-15R, IL-18R and IR-21R.As employed in this article, term " active NK receptor " does not comprise IL-2 receptor (IL-2R).Confirm whether the NK cell has activity or the method for whether breeding will be described in detail hereinafter, and be known those skilled in the art.

As it is employed in this article; Term " inhibition " or " " the NK receptor is meant any molecule on the NK cell surface to inhibition; When irriate; It causes and the active characteristic relevant, that the field is known of NK or active measurable minimizing, like the generation of cytokine (like IFN-γ and TNF-α), the increase of endocellular liberation calcium content or the ability of in the killing experiments that leads again, dissolving target cell that other place is in this manual described.The instance of such receptor comprises: KIR2DL1, KIR2DL2/3, KIR2DL4, KIR2DL5A, KIR2DL5B, KIR3DL1, KIR3DL2, KIR3DL3, LILRB1, NKG2A, NKG2C, NKG2E and LILRB5.Confirm the NK cell whether will describe in detail hereinafter by activated method, and be known those skilled in the art.

In the present invention; Term " block N K cell inhibiting property receptor or stimulate its activated receptor " is meant the ability of some chemical compound (being preferably antibody, its fragment or derivant), preferably is meant directly with at least a interaction like inhibition listed among KIR, NKG2A/C, NKp30, NKp44, NKp46 or other this paper or active NK cell receptor, or neutralizes the inhibition signal (under the situation of inhibition receptor) of autoreceptor or come the stimulus signal (under the situation of activated receptor) of autoreceptor.For the inhibition receptor, preferred chemical compound is preferably antibody or its fragment for can block the interactional chemical compound between HLA and the receptor.When this chemical compound was antibody, this antibody can be polyclonal antibody or be preferably monoclonal antibody.They can through hybridoma or through genetically engineered in order to express required variable region and the reorganization of constant region (body) cell produces.This antibody can be single-chain antibody or keep this antigenic specificity and like the hinge region of Fab fragment, Fab ' 2 fragments, CDR and these even lower levels of ScFv or other antibody derivatives of its variable region.These can be multipurpose antibody, reorganization (body) antibody, humanized antibody or their variant.

In context of the present invention, " common determinant " be meant determinant that a plurality of members by one group of associated receptor such as people KIR2DL receptor group have or antigen determining part (bunch).This determinant or antigen determining part (bunch) can show as the fragments of peptides that has by said member or conformation antigen determining part (bunch).In a particular embodiment, this common determinant comprise the antigen determining part discerned by monoclonal antibody DF200, NKVSF1 or EB6 (bunch).

In context of the present invention; Term antibody is meant that with a common determinant " combination " antibody has combining of specificity and/or affinity with described determinant, and for example being positioned at lip-deep other incoherent conformation of NK cells of human beings or determinant or structure is not bonded through high-affinity or specificity basically.More specifically, according to the present invention monoclonal antibody to the combination of said determinant can with said antibody and other antigen determining part or determinant combine make a distinction.

Thereby can be incorporated into NK cell inhibiting property receptor and stop the chemical compound of its stimulation to become " neutralization " or " inhibition " chemical compound; Be preferably antibody; Their blocking-up on this meaning, part is blocked the inhibition signal path that is mediated through NK cell inhibiting property receptor (like KIR or NKG2A/C receptor) at least.What is more important, this inhibitory activity can show for the KIR or the NKG2A/C receptor of several types, thus these chemical compounds (being preferably antibody) can be used for various patients, and have very high curative effect.

Term " reorganization (body) "; When being used for like cell or nucleic acid, albumen or carrier; Be meant this cell, nucleic acid, albumen or carrier through introducing external source nucleic acid or albumen or modified or this cell-derived cell of hanging oneself and revising like this through the natural acid transformed or albumen.Thereby for example, the gene of reorganization (body) cellular expression finds in the cell of natural (non-reorganization) form, or the natural gene of expressing should reach or do not express fully for unconventionality expression, low scale.

In context of the present invention, patient or patient comprise various mammalian subject or patient, more preferably human patients or patient.

Therapeutic antibodies

The present invention relates to the use of uniting of NK cell potentiating compounds and therapeutic antibodies.In the large therapeutic mass antibody any one may be used to the present invention.Basically, no matter any antibody be " exposed " or bonded with radioactive marker, toxin or other part (moiety), or no matter be total length or fragment, or no matter be that the modified derivative of true antibody or antibody can use.Preferably; This method is to be used to strengthen therapeutic efficiency; Therein the NK cell activity to the curative effect of institute's administering therapeutic property antibody play a role-but need not to be unique, and preferred antibodies or fragment should comprise naturally or after revising, comprise people Fc zone or can be by other zone of people Fc receptor (like Fc γ receptor) specific antibody identification.

Chemical compound of the present invention can be used to strengthen therapeutic antibodies and eliminate expression by the ability of the antigenic target cell of this therapeutic antibodies specific recognition.Correspondingly, part is by can being treated with the method for describing among this paper as any disease that cell caused or worsened or the disease of target by therapeutic antibodies at least.The specific examples of target cell comprises: the immunologically competent cell of the tumor cell relevant with allergy, autoimmune disease, allogeneic reaction etc., the cell of viral infection, homogeneous variant cell, morbid state (like bone-marrow-derived lymphocyte, T lymphocyte, present antigenic cell etc.), or or even healthy cell (like the endotheliocyte in the angiogenesis inhibitor therapeutic scheme).In context of the present invention, most preferred target cell is the cell of tumor cell and viral infection.This therapeutic antibodies can, for example, mediated cell toxic action or cytolysis are particularly through the cell-mediated cytotoxic effect (ADCC) of antibody dependent type.

ADCC need be used for the leukocyte receptors (Fc γ R) of the Fc part of IgG, and its function combines IgG sensitization+antigen with the cytotoxic cell that has Fc γ R, and in order to start cell activation mechanism.Therefore, this therapeutic antibodies can form immune complex.For example, immune complex can be by the tumor target spot of therapeutic antibodies covering.More specifically, this antibody can combine through CD16, and preferably the Fc zone through it combines.Confirm whether therapeutic antibodies combines with the such Fc γ receptor of CD16 and can use any suitable method to test; For example through confirming and CD16 polypeptide or its segmental combination of recombinating and producing; Optional being fixed on the carrier (supporter), or for example through confirming this therapeutic antibodies and known expression CD16 or suspecting the combining of cell of its expression CD16.

This therapeutic antibodies can be polyclone or be preferably monoclonal.They can express required variable region and the reorganization of fixed area (body) cell produces by hybridoma or through genetically engineered being used to.This antibody can be single-chain antibody or remain with antigenic specificity and other antibody derivatives of rudimentary hinge region, or their variant.These can be multipurpose antibody, recombinant antibodies, humanized antibody, their fragment or variant.Described its fragment or derivant preferentially are selected from Fab fragment, Fab ' 2 fragments, CDR and ScFv.Preferred fragment is a Fab.The therapeutic antibodies that comprises antigen fragment also can include but not limited to bi-specific antibody; A kind of example of suitable bi-specific antibody is to comprise antigen binding domain and to a tumor antigen special antigen binding domain special to CD16.Other the how segmental antibody formation that comprises comprises that the land with two different antibodies is attached to reorganization (body) bi-specific antibody derivant on the single polypeptide, is also referred to as BiTE ^TM(Kufer P, et al TRENDS in Biotechnology 2004; 22 (5): 238-244; With Baeuerle et al, Current Opinion in Molecular Therapeutics 2003; 5 (4): 413-419, the content of its disclosure is incorporated among this paper through quoting as proof.)

Therapeutic antibodies has specificity to surface antigen (for example membrane antigen) usually.Most preferred therapeutic antibodies is that tumor antigen (molecule of for example being expressed by tumor cell specific) like CD20, CD52, ErbB2 (or HER2/Neu), CD33, CD22, CD25, MUC-1, CEA, KDR, α V β 3 etc., is particularly had specificity to lymphoma antigen (for example CD20).This therapeutic antibodies has preferred IgG1 or IgG3Fc part for people or non-human primates, more preferably human IgG1.

In one embodiment, this antibody is included in the modification of its Fc part, and they strengthen the interaction of antibody and NK cell when ADCC.Generally include preferred modification in the Fc zone through the therapeutic antibodies of modifying (" through the antibody that changes ") like this, they can improve the binding affinity of this antibody and one or more Fc γ R.Being used to change antibody is being known in the art for the associated methods of the change of one or more Fc γ R; Can be referring to like the PCT application publication number being WO 2004/016750 (international application no is PCT/US2003/025399), WO 99/158572, WO 99/151642, WO98/123289, WO 89/107142, WO 88/107089 and United States Patent (USP) the 5th; 843, No. 597 and the 5th, 642; No. 821, their full content is incorporated among the application through quoting as proof.

The therapeutic antibodies of confirming among the present invention; As be used to treat rheumatic arthritis D2E7 (Cambridge Antibody Technology Group, plc (Cambridge, UK)/BASF (Ludwigshafen; Or sharp former times monoclonal antibody (the Infliximab) (Centocor of English Germany)); Inc., Malvern, PA; Be used for treating clone disease (Crohn ' s disease) and rheumatic arthritis) or the antibody that in International Patent Application PCT/US2003/025399 (full content is incorporated in the application as a reference with the list of references mode), discloses can be used in the method for instructing in the context of present patent application and modify or improve, and be used for treating the disease of typical these Antybody therapies of use.In certain embodiments, the invention provides the antibody through changing, they have altered affinity (higher or lower affinity) for active Fc γ R (being Fc γ RIII).In some preferred embodiment, provide for Fc γ R to have the more antibody through changing of high-affinity.Preferably such modification also has effector (effector) function of the Fc-mediation of change.

The modification that influences effector (effector) function of Fc-mediation be known in the field (referring to, like United States Patent (USP) the 6th, 194, No. 351, its full content is incorporated among this paper through quoting as proof).The aminoacid that can modify includes but not limited to: proline 3 29, proline 3 31 and lysine 322.Proline 3 29 and/or 331 can preferably be replaced by alanine with lysine 322, yet, also can consider to use other aminoacid replacement.Referring to No. the 6th, 194,551, International Publication No. WO 00/142072 and United States Patent (USP), its full content is incorporated among this paper through quoting as proof.

Therefore, the modification in Fc zone can comprise one or more changes that the aminoacid at the antibody Fc area discover is carried out.Such change can cause antibody to have improved antibody-mediated effector (effector) function, the improved binding ability of other Fc receptor (like the Fc active acceptor), active, the improved Clq of improved ADCC are combined activity, improved complement dependent form cytotoxic activity or their combination in any.

In one embodiment, this antibody is by the identification of Fc γ receptor-specific, and such Fc γ receptor such as FCGR3A (are also referred to as CD16, FCGR3, immunoglobulin G Fc receptor II I; IGFR3, for the segmental receptor of the Fc of IgG, low affinity IIIa; Referring to like OMIM146740), FCGR2A (be also referred to as CD32, CDw32, for the segmental receptor of the Fc of IgG, low affinity IIa, FCG2, immunoglobulin G Fc receptor II; Referring to like OMIM 146790); FCGR2B (be also referred to as CD32, for the segmental receptor of the Fc of IgG, low affinity IIb; FCGR2B, FC-γ-RIIB; Referring to like OMIM 604590), FCG1RA (is also referred to as CD64; For the segmental receptor of the Fc of IgG, high-affinity Ia; IGFR1; Referring to like OMIM 146760; The FCGR1 fragment of IgG, high-affinity Ic, immunoglobulin G Fc receptor IC, IGFRC; Referring to like OMIM 601503); Or FCGR1B (be also referred to as CD64, for the segmental receptor of the Fc of IgG, high-affinity Ib; Immunoglobulin G Fc receptor IB; IGFRB; Referring to like OMIM 601502).

The typical therapeutic antibodies of the present invention is that Rituximab (rituximab), Ah coming organize monoclonal antibody (alemtuzumab) and Herceptin (trastuzumab).These antibody can use according to the clinical standard of ratifying to be used for human patients.Other particular example of therapeutic antibodies comprises: for example, and the sharp former times monoclonal antibody (infliximab) of anti-CD22 monoclonal antibody (epratuzumab), basiliximab (basiliximab), Dary pearl monoclonal antibody (daclizumab), Cetuximab (cetuximab), labetuzumab, sevirumab (sevirumab), tuvurimab, palivizumab (palivizumab), English, Ao Mazuo monoclonal antibody (omalizumab), pearl monoclonal antibody (efalizumab), natalizumab (natalizumab), clenoliximab (clenoliximab) etc. in accordance with the law.Alternatively; When the chemical compound of the activated receptor that stimulates the NK cell is cytokine; This therapeutic antibodies is the antibody except that Rituximab (rituximab) or Trastuzumab (herceptin), and perhaps optional is the antibody except that anti-CD 20 or anti--HER2/ nerve sheath (neu) antibody.According to the present invention, other example of preferred therapeutic antibodies comprises: (this KIR receptor is at Carrington and Norman for anti--ferritin antibody (No. the 2002/0106324th, U.S. Patent application), anti--p140 and anti--sc5 antibody (WO02/50122) and anti--KIR (killer cell inhibition receptor) antibody The KIR Gene Cluster, describe among the May 3,2003, can obtain in following network address: Http:// www.ncbi.nlm.nih.gov/books), the content that above-mentioned each document is disclosed is incorporated among this paper through quoting as proof.During other example of therapeutic antibodies is listed in the table below, wherein any one (with other kind) may be used to this method.Should understand; Described in no matter whether being listed in the table below or in other place of this description; Any antibody that can eliminate target cell; Preferably through ADCC, all can be benefited, and 1 pair of wherein listed antibody of following table, listed target or the expression explanation of antibody all are not exhaustive from this method.

Table 1: therapeutic antibodies

Chemical compound is regulated the NK cytoactive

The NK cytoactive is to regulate through the mechanism of complicacy, and it relates to stimulus signal and suppresses signal.Correspondingly, effectively the cell-mediated treatment of NK both can through stimulate these cells also can through in realize with the inhibition signal.Should be noted that and anyly have blocking-up, suppress or other activity or the chemical compound of expressional function of activated receptor of chemical compound or activation, stimulation or other promotion NK cell of downward adjusting NK cell inhibiting property receptor acting all is operable.This comprises the chemical compound that can be incorporated into the NK cell receptor and directly block or stimulate them, like cytokine and micromolecule, polypeptide, and antibody.Be to be further noted that the mechanism that receptor is blocked or stimulated is not most important for advantage provided by the present invention.For example; This chemical compound can increase the expression of activated receptor or suppress the inhibition receptor expression; This chemical compound can stop the interaction between part and the inhibition receptor or strengthen the interaction between part and the activated receptor, and perhaps this chemical compound can directly be attached on the receptor and suppress their (under situation of inhibition receptor) or activate their (under situation of activated receptor).Important parameters is this chemical compound is eliminated the target cell ability in vivo to therapeutic antibodies effect.

Inhibition receptor on any NK cell surface can be as the target of chemical compound of the present invention.The NK cell is born adjusting (Karre et al., 1986 through major histocompatibility complex (MHC) type I-special inhibition receptor;

et al., 1989; The content of its disclosure is incorporated among this paper through quoting as proof).These specific receptors are incorporated into the polymorphic determinant of major histocompatibility complex (MHC) type I molecule or HLA, and suppression of natural killer (NK) cytolysis.In human body, be called the receptor family identification HLA type I allele group of killer cell Ig-appearance receptor (KIRs).

A plurality of KIR receptor groups are arranged, comprise KIR2DL, KIR2DS, KIR3DL and KIR3DS.KIR receptor identification HLA-C allotype with two Ig domains (KIR2D): KIR2DL2 (called after p58.1 in the past) or the gene outcome KIR2DL3 identification epitope shared that is closely related with group 2HLA-C allotype (Cw1,3,7 and 8), opposite KIR2DL1 (p58.2) the identification epitope shared with complementary (reciprocal) group 1HLA-C allotype (Cw2,4,5 and 6).Shown that by KIR2DL1 identification there is lysine (Lys) residue in HLA-C allelic the 80th (site).The identification of KIR2DL2 and KIR2DL3 is illustrated in the 80th (site) and has agedoite (Asn) residue.The very important point is that most of HLA-C allele have agedoite (Asn) or lysine (Lys) residue in the 80th (site).A KIR with 3 Ig domains, KIR3DL1 (p70) discerns an epitope of sharing with HLA-Bw4 allele.At last, a kind of homodimer molecule KIR3DL2 (p140) identification HLA-A3 and HLA-A11 with three Ig domains.

Although KIR and other type-I inhibition receptor (Moretta et al, 1997; Valiante et al, 1997; Lanier, 1998; The content of its disclosure is incorporated among this paper through quoting as proof) can be by NK cell co expression; But in the NK of any particular individual express spectra (repertoire); The cell of expressing independent KIR is arranged, so the only cell blocking-up of one type of specificity type I allele group being expressed of corresponding N K cell.Correspondingly, as mentioned below, when inhibition receptor during as target, this method will often relate to use with a plurality of inhibition receptors as target chemical compound, thereby guarantee to influence widely the NK cell that reaches maximum magnitude.

In certain embodiments; This chemical compound; Be preferably antibody or its fragment, block N K cell inhibiting property receptor, the inhibition signal of neutralization at least one optional inhibition receptor from comprise KIR2DL2, KIR2DL3, KIR2DL1, KIR3DL1, KIR3DL2, NKG2A and NKG2C one group.More preferably, the chemical compound of this block N K cell inhibiting property receptor is preferably antibody or its fragment, be in the chemical compound of the inhibition signal of KIR2DL2, KIR2DL3 and/or KIR2DL1, be preferably antibody or its fragment.

The present invention also can consider to use the combination of compounds of the different inhibition receptors of multiple block N K cell, and this chemical compound is preferably antibody or its fragment.Preferably; The chemical compound of block N K cell inhibiting property receptor is a species specific inhibition receptor optional from comprise KIR2DL1, KIR2DL2, KIR2DL3, KIR3DL1, KIR3DL2, NKG2A and NKG2C one group; And the cytotoxicity inhibitory action of the NK cell of KIR-that can suppress to be correlated with or NKG2A/2C-mediation, this chemical compound is preferably antibody or its fragment.For example, the chemical compound of block N K cell inhibiting property receptor can comprise a kind ofly having specific antibody for KIR2DL and have specific antibody with another kind of for KIR2DL2 and/or KIR2DL3.More preferably, the combination of compounds of this block N K cell inhibition receptor can suppress the cytotoxicity inhibitory action of the NK cell of KIR2DL1-, KIR2DL2-and KIR2DL3-mediation.In other embodiments, can use one or more inhibition receptors one or more chemical compounds as target, and with one or more activated receptors as target the intermixture of one or more chemical compounds.

For example, demonstrated for the specific monoclonal antibody of KIR2DL1 and can block KIR2DL1 Cw4 (or similarly) allele (Moretta et al., 1993; The content of its disclosure is incorporated among this paper through quoting as proof).In another example, the monoclonal antibody of anti-KIR2DL2/3 also has been described and can have blocked KIR2DL2/3HLACw3 (or similarly) allele (Moretta et al., 1993).Anti-NKG2A antibody also demonstrated can block N KG2A and HLA-E between inhibition interact.

Alternatively, this antibody can be selected from: and GL183 (KIR2DL2, L3 can be available from Immunotech, France and B eckton Dickinson, USA); EB 6 (KIR2DL1 can be available from Immunotech, France and Beckton Dickinson, USA); AZ138 (KIR3DL1 can be available from Moretta et al, Univ.Genova, Italy); (KIR3DL2 can be available from Immunotech, France) for Q66; (NKG2A can be available from Immunotech, France) for Z270; P25 (NKG2A/C can be available from Moretta et al, Univ.Genova, Italy) and DX9, Z27 (KIR3DL1 can be available from Immunotech, France and Beckton Dickinson, USA).

One preferred aspect, the present invention use monoclonal antibody with and fragment and derivant, a plurality of KIR on wherein said antibody, fragment or derivant and the NK cell surface or the cross reaction of NKG2A/C receptor and their the inhibition signal of neutralizing.

In one embodiment, the present invention uses monoclonal antibody, and it combines the common determinant of people KIR2DL receptor and suppresses corresponding inhibition passage.Preferably, the present invention uses monoclonal antibody, and it combines lip-deep KIR2DL1 of NK cells of human beings and KIR2DL2/3 receptor, and suppresses the inhibitory action of the NK cell poison of KIR2DL1-and KIR2DL2/3-mediation.This antibody specificity ground suppresses the HLA-c molecule and is incorporated into KIR2DL1 and KIR2DL2/3 receptor.More preferably, this antibody promotes the NK cytoactive in vivo.Because KIR2DL1 and KIR2DL3 (or KIR2DL2) are enough to cover most HLA-C allotype; It is respectively group 1HLA-C allotype and group 2HLA-C allotype; Therefore such antibody can be used for being increased in the effect of most of human individual's therapeutic antibodies, normally about 90% or more human individual in.In such an embodiment; Submit on July 1st, 2004, name is called any antibody that the PCT patent application PCT/FR 04/01702 of " Composition and methods for regulating NK cell activity (being used for regulating the compositions and the method for NK cytoactive) " describes and can correspondingly be used for the present invention, the content of its disclosure is incorporated among this paper through quoting as proof.

In a specific purpose of the present invention, the antibody of this block N K cell inhibiting property receptor is monoclonal antibody, the common determinant of wherein said antibodies KIR2DL people receptor, and the inhibitory action of the NK cell cytotoxicity of inhibition KIR2DL-mediation.This antibody is incorporated into and the monoclonal antibody DF200 or the identical epitope of NKVSF1 that are produced by hybridoma DF200 or NKVSF1 respectively more specifically, and/or is incorporated into the lip-deep KIR receptor of NK cells of human beings with the monoclonal antibody DF200 or the NKVSF1 competition that are produced by hybridoma DF200 or NKVSF1 respectively.Like what discussed, whether the example of antibody, functional test and definite antibody are described in PCT patent application PCT/FR 04/01/01702 with the bonded test of said antibody competition.

In a particular embodiment, this monoclonal antibody is the monoclonal antibody DF200 that is produced by hybridoma DF200.In another embodiment, this monoclonal antibody is EB6, or this antibodies is in the epitope identical with monoclonal antibody EB6, or combines with monoclonal antibody EB6 competition.In other embodiments, this antibody is fragment or the derivant of antibody DF200 or EB6.Produce antibody DF200 hybridoma (CNCM culture collection) preservation, identifier (Identification no.) " DF200 ", registration number CNCM I-3224 at national microbial preservation center, register on June 10th, 2004; Collection Nationale de Cultures de Microorganismes; Institut Pasteur, 25, Rue du Docteur Roux; F-75724Paris Cedex 15, France.Antibody NKVSF1 can (Cergy Sainte-Christophe France) obtains catalogue label (Catalog ref no.) MCA2243 from Serotec.

In another embodiment of the present invention, the chemical compound that is used to strengthen efficiency of therapeutic antibodies stimulates the activated receptor of NK cell.Can use any activated receptor; Like NKp30 (referring to like PCT WO 01/36630; The full content of its disclosure is incorporated among this paper through quoting as proof), NKp44 (referring to like Vitale et al. (1998) J.Exp.Med.187:2065-2072, the content of its disclosure being incorporated among this paper through quoting as proof), NKp46 be (referring to like Sivori et al. (1997) J.Exp.Med.186:1129-1136; Pessino et al. (1998) J.Exp.Med.188:953-960; The content of its disclosure is incorporated among this paper through quoting as proof), NKG2D (referring to like OMIM 602892), IL-12R, IL-15R, IL-18R, IL-21R or activatory KIR receptor, for example the KIR2DS4 receptor (Carrington and Norman, The KIR Gene ClusterMay 3; 2003 can available from: http://www.ncbi.nlm.nih.gov/books); Or be present in any other antibody on the main fragment of NK cell, and its activity causes cell-stimulating or propagation, and preferably this cell was suppressed by the inhibition receptor as inhibition KIR receptor so in the past.This chemical compound can be any molecular entity, comprises polypeptide, micromolecule and antibody.The chemical compound of example comprises any natural, reorganization or synthetic part, and they can interact with activated receptor.For example a kind of chemical compound of the NK of stimulation cell activation receptor can be as with the interactional IL-12 of IL-12 receptor (IL-12R), with the interactional IL-15 of IL-15 receptor (IL-15R), with the interactional IL-18 of IL-18 receptor (IL-18R), with cytokine such as the interactional IL-21 of IL-21 receptor (IL-21R).Such chemical compound known from IL-12 (Research Diagnostics, NJ, DI-212), IL-15 (Research Diagnostics, NJ, RDI-215), IL-21 (Asano et al, FEBS Lett.2002; 528:70-6).The chemical compound of preferred stimulation NK cell activation receptor is the chemical compound except that IL-2.Other stimulate the exemplary compounds of NK cell activation receptor to comprise to combine the NK cell receptor, be selected from the antibody in the group of forming by NKp30, NKp44, NKp46, NKG2D, KIR2DS4 and other activatory KIR receptor.

In some preferred embodiment; This activated receptors is the nature cell toxicity receptor of on the NK cell, finding (NCR); Preferred NCR is selected from NKp30, NKp44 or NKp46, and the chemical compound of this stimulation activated receptor is to be incorporated into same epitope or to compete with it with any monoclonal antibody that is selected from AZ20, A76, Z25, Z231 and BAB281 to combine.

Can be incorporated into arbitrary NK cell receptor described herein through utilizing in the various standard methods any one to measure arbitrary chemical compound.For example, colorimetric ELISA-categorical measures method can be used to measure immunoprecipitation and radioimmunoassay.Can use the competition test; For example compare the binding ability that testing compound and known compound are incorporated into the NK cell receptor; Wherein contrast (for example BAB281, its specificity is incorporated into NKp46) and testing compound are through mixing (or pre-absorption) and being applied in the sample that contains the albumen (being NKp46 under like the situation at BAB281) that comprises epitope.Experimental technique based on ELISA (ELISA), radioimmunoassay, western blotting (Western blotting) and BIACORE is applicable to this simple competition research, and is well known in the art.

The inhibiting inhibition of cytotoxicity for the NK cell of KIR-or NKG2A/C-mediation; Or can measure with various checks or test for the stimulation of the activation NK cell of NKp30, NKp44, NKp46 or NKG2D-mediation, as combining test, cell toxicant test or other molecular testing or cell tests.

In a kind of specific variant, inhibitory activity through said chemical compound (being preferably antibody) on the positive target of HLA-C or HLA-E respectively reconstruct KIR or the positive NK clone's of NKG2A/C solvability explain.In another particular embodiment; This chemical compound (being preferably antibody) is incorporated into KIR2DL1 with inhibition HLA-C molecule and KIR2DL3 (or the KIR2DL2 that is closely related) receptor limits; More preferably, change the HLA-C molecule that is selected from Cw1, Cw3, Cw7 and Cw8 (or being selected from the HLA-c molecule that has agedoite (Asn) residue at the 80th) with it and be incorporated into the ability of KIR2DL2/3 and limit the ability that the HLA-C molecule that is selected from Cw2, Cw4, Cw5 and Cw6 (or being selected from the HLA-c molecule that has lysine (Lys) residue at the 80th) is incorporated into KIR2DL1.

The inhibitory activity of chemical compound of the present invention (being preferably antibody) or enhanced activity; Can measure with in numerous methods any one; For example through as the effect of free calcium ions outside described its pair cell of Sivori et al. (1997) J.Exp.Med.186:1129-1136, the content of its disclosure is incorporated among this paper through quoting as proof.The NK cytoactive can utilize also that test detects based on the cytotoxicity of cell, for example measures the release of chromium, stimulates NK cell killing such as P815, K562 or other as at Sivori et al. (1997) J.Exp.Med.186:1129-1136 as measuring antibody; Vitale et al. (1998) J.Exp.Med.187:2065-2072; Pessino et al. (1998) J.Exp.Med.188:953-960; Neri et al. (2001) Clin.Diag.Lab.Immun.8:1131-1135; The ability of the target cell that suitable tumor cell disclosed in Pende et al. (1999) J.Exp.Med.190:1505-1516 is such is incorporated in the full content of its disclosure among this paper through quoting as proof.Also provided suitable cell toxicant test in the embodiment of description of the present invention part.In a preferred embodiment, this antibody causes the enhancing of NK cell toxicant at least 10%, preferably causes the enhancing of NK cell toxicant at least 40% or 50%, or more preferably causes the enhancing of NK cell toxicant at least 70%.

The NK cytoactive also can describe with cytokine-release test, wherein NK cell and antibody incubation is produced (the for example generation of IFN-γ and TNF-α) with the cytokine that stimulates the NK cell.In an exemplary scheme, the IFN-γ that is produced by PBMC measures with colouring method in cell surface and the kytoplasm, and after cultivating 4 days, analyzes with the flow cytometry method.Briefly, be that 5 μ g/ml add and cultivated at least 4 hours with brefeldin A (Brefeldin A, Sigma Aldrich) with final concentration.Then, at permeability effect (IntraPrep ^TMBeckman Coulter) before, this cell is hatched with the monoclonal antibody (mAb) of anti--CD56 with anti--CD3, and resist-IFN-γ or PE-IgG1 (Pharmingen) dyeing with PE-.Can use ELISA (GM-CSF:DuoSet Elisa, R&D Systems, Minneapolis, MN by the GM-CSF of the active NK cell generation of polyclone and the amount of IFN-γ; IFN-γ: OptE1A set, Pharmingen) measure supernatant and obtain.

In a preferred embodiment, measure the ability to function of antibody,, show that this chemical compound is applicable to the present invention when the ability that can activate NK cells of human beings is the same with the ability that activates non-NK cells of human beings at least when good to activated NK cells of human beings.Especially can measure chemical compound and be used to strengthen therapeutic antibodies in vivo or the external ability of directly eliminating the target cell that (exhaustions) suit through the NK cell.

Chemical compound of the present invention (being preferably antibody) can have part inhibition or part stimulating activity, and for example part reduces the inhibitory action of the NK cell poison of KIR2DL-mediation, or part activates the NK cell through stimulating NCRs or other receptor to some extent.The preferred chemical compounds of great majority can both suppress the NK cytoactive of (or under the situation of activated receptor, stimulating) at least 20% (being preferably at least 30%, 40% or 50% or higher), compare like the cell during with this chemical compound not in cell toxicant is measured.Preferably, this chemical compound can also make and eliminate the target cell degree and improve 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, 1000% or more when not having this chemical compound.Alternatively optional, the preferred chemical compound of the present invention (being preferably antibody) can be induced coupling or that HLA is compatible or from somatic target cell crowd's dissolving, promptly can not be by the effective dissolved cell mass of NK cell when not having said antibody.Correspondingly, chemical compound of the present invention also can be used and promote NK cell activity in vivo to limit.

The present invention also can comprise such embodiment; Its moderate stimulation NK cell activation receptor or the chemical compound of preferably blocking its inhibition receptor are the monoclonal antibody fragments with substantially the same antigenic specificity, and it includes but not limited to Fab fragment, Fab ' 2 fragments, CDR and ScFv.And, this monoclonal antibody can be humanized, the people's or chimeric (the for example antibody of bispecific or functionalization).Though stimulating the antibody of activated receptor also can be fragment, total length preferably.Derivant (for example have modification sequence or have bonded assorted source functional group or other chemical compound) can be used for any antibody described herein.

According to the present invention, block N K cell inhibition receptor or stimulate the antibody of its activated receptor to prepare with various technical methods well known in the art.Normally through with comprising the polypeptide of KIR, NKG2A/C, NCR (for example NKp30, NKp44, NKp46) or NKG2D or the immune original immune non-human animal of the immunogen fragment of these polypeptide arbitrarily, and collect that splenocyte (producing hybridoma through fusion with suitable cell line) prepares.From each species produce monoclonal antibody method be known in those skilled in the art (referring to, like Harlow et al., " Antibodies:A laboratory Manual " CSFH Press, 1988; Goding, " Monoclonal Antibodies:Principles and Practice " Academic Press, 1986; The content of its disclosure is incorporated among this paper through quoting as proof).More specifically, these methods comprise with antigen comes immune non-human animal, reclaims splenocyte subsequently, with the cell that obtains immunity (immortalization), merges like the myeloma cell then.The hybridoma that obtains produces monoclonal antibody, and selects in order to separate independent clone through restricted dilution.Also can produce antibody through selecting the immunoglobulin combinatorial library, for example Ward et al (1989) is said; The content of its disclosure is incorporated among this paper through quoting as proof.

According to the present invention, preferred block N K cell inhibiting property receptor or the antibody that stimulates its activated receptor through with comprise like the KIR2DL polypeptide, more preferably the immunogen immune for the activity of people KIR2DL polypeptide or inhibition NK cell receptor prepares.This KIR2DL polypeptide can comprise full length sequence or its fragment or the derivant of people KIR2DL polypeptide, and typical immunogen fragment promptly comprises the part of the polypeptide of antigen determining part being preferably T or B cell antigen deciding section.Such fragment contains at least 7 successive aminoacid in its mature polypeptide sequence usually, is more preferably its 10 successive aminoacid at least.They come the extracellular domain of autoreceptor basically.In a preferred embodiment, this immunogen comprises the polypeptide in wild type people KIR2DL, NCR or other adipose membrane, is usually located at the surface of cell.In a certain embodiments, this immunogen comprises complete NK cell, particularly complete NK cells of human beings, and optional is handled or dissolved.

Though therapeutic antibodies can have through the Fc zone of modifying so that strengthen their combinations through the receptor as CD16; But the enhanced antibody of NK cell will have the Fc district of change so that reduce its affinity to the Fc receptor in certain embodiments; Thereby reduce the NK cell by the probability of antibodies, this will cause himself to be combined and dissolve.

The antibody of the KIR2DL receptor of block N K cell can be in order to the preparation of below method, and this method comprises: i) with the immune original immune non-human mammal that comprises the KIR2DL polypeptide; Ii) prepare monoclonal antibody by said animal through immunity, wherein said monoclonal antibody combines said KIR2DL polypeptide; Iii) from step I i) select the monoclonal antibody with the KIR2DL polypeptide cross reaction of at least two different serotypes; And the monoclonal antibody of (c) that iv) select to suppress the NK cyto-inhibition of KIR2DL-mediation.

Step (iii) and order (iv) can change.Alternatively, be described below, this method may further include the additional step of making this monoclonal antibody fragment or derivant.

In another variant, this method comprises: i) by monoclonal antibody or its fragment or the derivant of selecting a kind of KIR2DL polypeptide cross reaction of and at least two different serotypes in library or the express spectra; And from step I) select the antibody of the NK cell inhibiting effect that suppresses the KIR2DL-mediation.

Should be noted that any one of these methods may be used to select that the NK cell receptor of any kind of (inhibition or activity) of total one or more epitopes is had specific antibody or antibody fragment.For example, similarly method can be used to prepare the KIR3DL of block N K cell or the antibody of NKG2A/C receptor or stimulation NK cell activation receptor.

In the preferred embodiment, use in these methods, or the non-human animal who is used to produce any antibody as herein described is mammiferous, like Rodents (like mice, rat etc.), cattle, pig, horse, rabbit, goat, sheep etc.

In addition, any antibody described herein can be through genetic modification or genetically engineered to be applicable to the mankind, like humanized antibody, chimeric antibody or people's antibody.The method that is used for humanized antibody is known in the field.Generally speaking, humanized antibody according to the present invention has one or more amino acid residues of being introduced by original antibody.These muroids or other non-human amino acid residue are commonly referred to as " introducing " residue, and they are normally from " introducing " variable region.Humanization can be realized (Jones et al. (1986) Nature 321:522 through the following method of implementing Winter and colleague basically; Riechmann et al. (1998) Nature 332:323; Verhoeyen et al. (1998) Science 239:1534 (1998)).In some cases, such " humanization " antibody is chimeric antibody (No. the 4th, 816,567, Cabilly et al. United States Patent (USP)), and wherein at least one complete people variable region is replaced by the corresponding sequence from original antibody basically.In fact, humanized antibody according to the present invention is typical people's antibody, number of C DR residue is wherein arranged and have some FR residues to be replaced by the residue from the similar site in the original antibody.

The another kind of method of making " humanization " monoclonal antibody is with

(Abgenix; Fremont is CA) as being used for mice immunized.XenoMouse is the muroid host who has by the substituted immunoglobulin gene of functional human immunoglobulin gene.Therefore, the antibody by the B cell manufacturing of this Mus that is produced by this Mus or its hybridoma is by humanized.XenoMouse is at United States Patent (USP) the 6th, 162, describes in 963, and the full content of its disclosure is incorporated among this paper through quoting as proof.A kind of similar method can be utilized HuMAb-Mouse ^TM(Medarex) realize.

People's antibody also can produce with various other immunological techniques; As use other can the expressing human antibody expression spectrum by genetically engineered transgenic animal (Jakobovitz et al.; Nature 362 (1993) 255), or with phage explicit representation selection antibody expression spectrum.This technology is known to those skilled in the art, and can be come into effect by the monoclonal antibody that the present invention discloses.

Antibody of the present invention also can be generalized to " chimeric " antibody (immunoglobulin); Consistent or the homology of corresponding sequence in the part of heavy chain and/or light chain and the original antibody wherein; And the remainder of chain with from the antibody of other kind or belong to another antibody class or the consistent or homology of segmental corresponding sequence of subclass and these antibody; As long as they demonstrate required BA (Cabilly et al., supra; Morrison et al., (1984) Proc.Natl.Acad.Sci.81:6851).

Be to be further noted that when this block N K cell inhibition receptor or when stimulating the chemical compound of NK cell activation receptor to be antibody, this antibody can be polyclone or monoclonal preferably.This antibody can or be expressed required variable region and constant region through genetically engineered recombinant cell by hybridoma and obtained.This antibody can be single-chain antibody or other antibody derivatives or its variant that keeps antigenic specificity and rudimentary hinge region.This antibody can be multipurpose antibody, recombinant antibodies, humanized antibody or its fragment or derivant.Its said fragment or derivant preferentially are selected from Fab fragment, Fab ' 2 fragments, CDR and ScFv.Preferred fragment is a Fab.The antibody that comprises antibody fragment also can include but not limited to bi-specific antibody.Instance is to comprise one to the specific antigen land of activated receptor and bi-specific antibody to the specific antigen land of tumor antigen (disclose WO No. 01/71005 referring to the PCT application, the content of its disclosure is incorporated among this paper through quoting as proof).

Compositions with use (administration)

The present invention relates to a kind of compositions; Comprise at least a block N K cell inhibition receptor or stimulate the chemical compound (being preferably antibody or its fragment) of its activated receptor; With a kind of therapeutic antibodies; Use said composition be used to improve this therapeutic antibodies curative effect, be used to improve with the intravital ADCC of patient of therapeutic antibodies treatment or be used to treat patient in spite of illness; Especially need to eliminate (exhaustion) disease as the cell (being preferably the cell of morbid state) of target like cell, cancerous cell or other pathogenic cell of viral infection.Preferably, this disease is selected from cancer (disease), autoimmune disease, inflammation, viral disease.This disease also relates to transplant rejection, more preferably allograft rejection and graft versus host disease (GVHD).

More specifically, this treatment of diseases need be eliminated the cell as target, is preferably the diseased cells cell, tumor cell or other pathogenic cell like viral infection.Preferably, this disease is cancer, infectiousness or immunological diseases.More preferably, this disease is selected from the group of being made up of cancer, autoimmune disease, inflammation, viral disease.This disease also relates to transplant rejection, more preferably allograft rejection and graft versus host disease (GVHD).

Said disease comprises the superfluous natural disposition propagation (neoplastic proliferation) of hematopoietic cell.Alternatively, said disease can be selected from the one group of disease that comprises lymphoid leukemia, acute or chronic lymphocytic leukemia, Hodgkin lymphoma, non-Hodgkin lymphoma, myelodysplastic syndrome, multiple myeloma and chronic lymphocytic leukemia.Said disease also comprises otorhinolaryngology (ENT) cancer, colorectal carcinoma, breast carcinoma, epithelial cancer.Said disease comprises that cytomegalovirus (CMV) infects and hepatitis B.Said disease comprises clone disease, rheumatic arthritis, asthma, psoriasis (psoriasis), multiple sclerosis or diabetes.Especially can treat any disease cited in the preamble table.

Said therapeutic antibodies can combine through CD16, preferably combines through its Fc zone.Preferably, said therapeutic antibodies has human IgG1 or IgG3 Fc part, especially monoclonal antibody or its fragment, more preferably people's antibody, humanized antibody or the chimeric antibody as Rituximab, or its fragment.

At least a combination the in said block N K cell inhibition receptor or the chemical compound (being preferably antibody or its fragment) that stimulates its activated receptor and KIR, KNG2A/C, NCR or the NKG2D people's receptor; And or suppress the Cytotoxic inhibitory action of the NK cell of relevant KIR2DL, KIR3DL and/or NKG2A/C-mediation, perhaps stimulate the Cytotoxic activation of the NK cell of relevant NCR or NKG2D-mediation.In a preferred embodiment; Used KIR2DL people's receptor; For example be selected from the lineup's receptor that comprises KIR2DL1, KIR2DL2, KIR2DL3, or used KIR3DL people's receptor, for example be selected from the lineup's receptor that comprises KIR3DL1 and KIR3DL2.

The Cytotoxic inhibitory action of the NK cell of the KIR2DL-mediation that in a preferred embodiment, this NK cell potentiating compounds at least one KIR2DL people's receptor of combination and inhibition are relevant.Preferably, this KIR2DL people's receptor is selected from the lineup's receptor that comprises KIR2DL1, KIR2DL2, KIR2DL3.In a preferred embodiment, this chemical compound is preferably antibody or its fragment, in conjunction with the common determinant of KIR2DL people's receptor and suppress the Cytotoxic inhibitory action of the NK cell of KIR2DL-mediation.More preferably, said chemical compound is preferably antibody, in conjunction with the common determinant of KIR2DL1, KIR2DL2, KIR2DL3 people's receptor and suppress the Cytotoxic inhibitory action of the NK cell of KIR2DL1-, KIR2DL2-, KIR2DL3-mediation.In a particular embodiment; Said chemical compound (being preferably antibody) is suppressed at the 80th the HLA-C allele molecule with lysine (Lys) residue and is incorporated into people KIR2DL1 receptor, and is incorporated into people KIR2DL2 and KIR2DL3 receptor at the 80th the HLA-C allele molecule with agedoite (Asn) residue.In another particular embodiment, this antibodies in the identical epitope of monoclonal antibody DF200 that produces by hybridoma DF200.Can be selectively, this antibody is incorporated into the lip-deep KIR receptor of NK cells of human beings with the monoclonal antibody DF200 competition that is produced by hybridoma DF200.In a preferred embodiment, this antibody is the monoclonal antibody DF200 that is produced by hybridoma DF200.In another embodiment, this antibody competition combines or is incorporated into the epitope identical with monoclonal antibody EB6.

According to the present invention, said composition can comprise the different inhibition receptors of multiple inhibition NK cell and/or stimulate the combination of compounds of one or more activated receptors of NK cell, be preferably antibody or its fragment.Preferably; The chemical compound of block N K cell inhibition receptor; Be preferably antibody or its fragment; Inhibition receptor to being selected from KIR2DL1, KIR2DL2, KIR2DL3, KIR3DL1, KIR3DL2, NKG2A and NKG2C has specificity, and the inhibitory action of the NK cell cytotoxicity of KIR-that can suppress to be correlated with or NKG2A/C-mediation." combination of compounds can suppress the inhibitory action of the NK cell cytotoxicity of KIR2DL1-, KIR2DL2-and KIR2DL3-mediation in more preferably, " neutralization.Through combination of compounds is provided, can in the patient of maximum quantity, block the different inhibition receptors of maximum quantity.Equally; Can use stimulate the different activities chemical compound (or when the inhibition receptor exists; Be incorporated into the different epitopes in the single receptor) chemical compound combination, for example the combination in any that is selected from the group that NKp30, NKp44, NKp46 and NKG2D form two kinds or more kinds of receptors is produced together the chemical compound of activation.In addition, also can use the chemical compound that comprises one or more blocking-up inhibition receptors to stimulate the combination of compounds of activated receptor with one or more.Be described below, in a preferred embodiment, before using this method, can obtain a NK cell sample, and for example when having target cell and therapeutic antibodies, can measure of the reaction of NK cell for the different compounds combination from the patient.

Compositions of the present invention can comprise stabilizing agent, antiseptic of any medicine acceptable carrier or excipient, buffer commonly used, isotonic solution, waterborne suspension, optional interpolation etc.Dosage form commonly used comprises saline solution, and optional protection or stable molecule, like high-molecular-weight protein (for example human serum albumin).

Also can provide to comprise one or more therapeutic antibodies the combination in any of one or more NK cell potentiating compounds, and the test kit that preferably has their operation instruction.

According to the method for the invention and compositions, block N K cell inhibition receptor or stimulate the chemical compound of its activated receptor is preferably antibody or its fragment, and therapeutic antibodies with " effectively " or " effectively treatment " amount is used (giving).

The effective dose that is applied to receiver's therapeutic antibodies can be that about 0.1mg/kg is to about 20mg/kg.Yet the effective dose of antibody depends on the form of antibody (full Ig or segment), must measure the affinity and the pharmacokinetic parameter of monoclonal antibody (mAb) to every kind of specific antibody.

Block N K cell inhibiting property receptor or stimulate the chemical compound (being preferably antibody or its fragment) of its activated receptor to be applied to receiver's effective dose can be at about 0.1mg/kg between about 20mg/kg.Yet the effective dose of antibody depends on the form of antibody (full Ig or segment), must measure the affinity and the pharmacokinetic parameter of monoclonal antibody (mAb) to every kind of specific antibody.

In an important embodiment of the present invention, use chemical compound of the present invention to be issued to therapeutic effect in the condition of the therapeutic antibodies dosage that reduces.The use of therapeutic antibodies (for example dosage, administration prescription) maybe be owing to side effect be limited, and for example reduces, reaches other symptom using the heating, headache, asthma, the blood pressure that occur under the situation of Rituximab.Correspondingly; The NK cell potentiating compounds of describing among this paper can be co-administered in many patients with the therapeutic antibodies of standard dose (that is, the RD during no any other chemical compound), thereby strengthened the curative effect of standard dose in the patient who needs bigger curative effect originally; In other patients; For example those receive the patient that side effect has a strong impact on, and use this chemical compound and can make its condition at the therapeutic antibodies dosage that reduces be issued to therapeutic effect, thereby avoided side reaction.In fact; The NK cell potentiating compounds of optimal dosage skilled medical practitioner can determine this therapeutic antibodies and to(for) given patient with use prescription, for example this therapeutic scheme is for patient's special requirement and patient's the whole state of an illness and Yan Douhui is optimum.A large amount of lists of references can be used for instructing the optimal dose of decision therapeutic antibodies and NK cell potentiating compounds, Remington:The Science and Practice of Pharmacy for example, by Gennaro (2003), ISBN:0781750253; Goodman and Gilmans The Pharmacological Basis of Therapeutics, by Hardman, Limbird & Gilman (2001), ISBN:0071354697; Rawlins E.A., editor, " Bentley ' s Textbook of Pharmaceutics ", London:Bailliere, Tindall and Cox, (1997); And other document.

In one embodiment; The medical treatment practitioner can gradually reduce amount or dispenser dosage or the dispenser frequency with the co-administered given therapeutic antibodies of any one NK cell potentiating compounds of the present invention; And monitor the curative effect of this therapeutic antibodies, for example monitor the NK cytoactive, the target cell that exists in the monitored patient body; Monitor various clinical indices; Or through other method, and corresponding to the relative concentration or the dispenser pattern of monitored results adjustment of treatment property antibody and/or NK potentiating compounds, thereby make therapeutic effect best and suppress side effect.

In another group embodiment; Before administering therapeutic property antibody and NK cell potentiating compounds (and if suitable; During treating) will obtain the NK cell from the patient, and measure, in order to the definite ideal chemical compound that will use or the greatest treatment efficacy of chemical compound group.The inhibition that for example exists on the NK cell or the characteristic of activated receptor can be identified, and the chemical compound of being used its specifically to most important receptor.Alternatively optional, resulting NK cell can be hatched with therapeutic antibodies and target cell, and can measure the ability that one or more chemical compounds are eliminated in order to the intensifier target cell.No matter one or more chemical compounds is any strengthening external elimination (exhaustions) ability when the most effective, can be selected for this Therapeutic Method.

Chemical compound and/or therapeutic antibodies appropriate dosage generally also can be external or in animal model, confirm; For example external through the therapeutic antibodies of various concentration is hatched in the presence of target cell, NK cell (being preferably NK cells of human beings), optional other immunocyte; And change the concentration of one or more NK cell potentiating compounds, and be determined at degree and the speed that target cell is eliminated under the different condition with the assay method (for example like the described method of embodiment part) of standard.Alternatively; Change the dosage of the therapeutic antibodies that animal model gave of the disease of available antibodies (animal model that for example under the Rituximab situation, is used for NHL) treatment along with the dosage that changes chemical compound described herein, and measure the curative effect (for example confirming) of antibody in the treatment animal by suitable clinical, cell or molecular assay or standard.

Can directly inject the patient according to compositions of the present invention, generally be to inject through intravenous, intraperitoneal, intra-arterial, intramuscular or percutaneous approach.Some monoclonal antibodies demonstrate curative effect clinically, and like Rituxan (Rituximab) or Xolair (Ao Mazuo monoclonal antibody), and similarly dispenser (administration) scheme (like dosage form and/or dosage and/or application method) can be used for compositions of the present invention.

In addition, compositions of the present invention may further include or can be co-administered with other active agent or therapeutic scheme, as with chemotherapy or other immunotherapy, simultaneously or use subsequently.

In some preferred embodiments, method of the present invention further comprises one or many injection two or more block N K cell inhibition receptor or stimulates the chemical compound of its activated receptor.Therefore, these two or more chemical compounds can be united use.This can be used as the bigger increase that causes ADCC and therapeutic antibodies curative effect, and/or can be used as the dosage that reduces specific block N K cell inhibition receptor or stimulate the chemical compound of its activated receptor.When for example, known chemical compound as IL-2 increases the dosage use is virose.Therefore, it is a kind of in the method that the patient's interior therapeutic disease that needs is arranged that the present invention preferentially provides, and comprising: a) said patient being used at least two kinds of block N K cell inhibition receptors or stimulating the preferred of its activated receptor is antibody or its segmental chemical compound; And b) said patient is used a kind of therapeutic antibodies.For example; A kind of preferred scheme is that said two kinds of chemical compounds are that (i) first chemical compound is selected from the group of being made up of the antibody of the antibody that stimulates NCR or NKG2D receptor or activatory KIR receptor and blocking-up inhibition KIR receptor or NKG2A, and (ii) second chemical compound is selected from the group of being made up of IL-12, IL-15, IL-18 and Il-21.Therefore; It is a kind of in the method that the patient's interior therapeutic disease that needs is arranged that the present invention further provides, and comprising: a) according to the present invention said patient being used a kind of block N K cell inhibition receptor or stimulates the preferred of its activated receptor is antibody or its segmental chemical compound; And b) said patient is used a kind of therapeutic antibodies; And c) said patient is used IL-2.IL-2 can be from Research Diagnostics, NJ, RDI-202, or Chiron Corp. (Emeryville CA) obtains.

Can administer cytokines according to the application program of any suitable; And can be before using block N K cell inhibition receptor or stimulating the chemical compound of its activated receptor, simultaneously and/or use afterwards, and before administering therapeutic property antibody, simultaneously and/or use afterwards.In a typical example, this cytokine inject block N K cell inhibiting property receptor first or stimulate its activated receptor chemical compound on the same day by injection first, and dispenser every day in 5-10 days.Said method possibly comprise by subcutaneous route 1 or 2 times/day injection cytokines.

The dosage of cytokine selects to depend on the patient's condition of waiting to treat the patient.In a preferred embodiment, can use the cytokine of relative low dosage.For example, when the pharmaceutical composition that contains cytokine was used for the subcutaneous injection of every day, a of effective dose was usually less than the 100 ten thousand unit cell factor/m ²/ day.In a preferred embodiment, IL-2 with every day dosage be lower than 100 ten thousand units/m ²Subcutaneous injection 5-10 days.The more detailed situation of using cytokine is referring to open PCT/EP/0314716 of international patent application and U.S. Patent application the 60/435th; No. 344; Name is called " Pharmaceutical compositons having an effect on the proliferation of NK cells and a method using the same ", and the content of its disclosure is incorporated among this paper with way of reference.Should be noted that therapeutic antibodies and NK cell potentiating compounds can together use; For example injection simultaneously; Or can use simultaneously but with different dosage forms, or can use separately, for example several hours, a couple of days or several weeks are used this chemical compound before or after compound administration.

Disclose in further aspect of the present invention and the advantage test portion below, this is to be understood that to being to illustration of the present invention but not limiting the scope of the invention.

Embodiment

Embodiment 1: full KIR2DL production of antibodies

The generation of the purification of PBL and polyclone or clone NK cell line

PBL is through Ficoll Hypaque gradient method and discard the donor that attached cell (plastic adherent cell) derives from health.In order to obtain the NK cell of enrichment; PBL is hatched (4 ℃ following 30 minutes) with the monoclonal antibody (mAbs) of anti-CD3, anti-CD4 and anti-HLA-DR; Use sheep anti mouse magnetic bead (Dynal) (4 ℃ following 30 minutes) subsequently and carry out immunological magnetic bead sorting with methods known in the art (Pende et al., 1999).With CD3 deficient cells-(CD3 minus cell), CD4 deficient cells-, the DR deficient cells is at feeder cell that shone and 100U/ml interleukin-22 (interleukin II) (Proleukin; Chiron Corporation) and on the 1.5ng/ml phytohaemagglutinin A (Gibco BRL) cultivate, thereby obtain polyclone NK cell family.The NK cell is cloned through restricted dilution, and the clone of NK cell discerns through the flow cytometry method that is used for the cell surface receptor expression.

Following clone is used for this research:

CP11, CN5 and CN505 are the positive colonies of KIR2DL1, and by EB6 or XA-141 antibody staining.CN12 and CP 502 are positive colonies of KIR2DL3, and by the GL183 antibody staining.

The flow cytometry method is analyzed

Employed monoclonal antibody (mAb) is JT3A (IgG2a, anti-CD3), EB6 and the GL183 (being respectively anti-KIR2DL1 of IgG1 and anti-KIR2DL3) that in this laboratory, prepare, the anti-KIR2DL1 of XA-141IgM (with EB6, anti-CD4 (HP2.6), anti-DR (DR1.12, IgG2a) compares have identical specificity).Except JT3A, HP2.6, DR1.12, have that mutually homospecific commercially available the monoclonal antibody of the Beckman coulter company in Fu Ledun city can be used for embodiment from the California.EB6 and GL183 be can be from the California Beckman coulter company in Fu Ledun city be purchased.XA-141 is not commercially available, but EB6 can be used to be controlled at the dissolved reconstruct described in the document (Moretta et al., 1993).

The flow cytometry method

Cell with suitable antibody staining (4 ℃ following 30 minutes), is combined polyclone anti-mouse antibody (Southern Biotechnology Associates Inc) through PE or FITC subsequently.(Becton Dickinson, Mountain View CA) carry out analytical test through cell fluorescence determination and analysis method on FACSAN equipment with sample.

Cytotoxicity experiment

NK clone's cell lysis activity detects through 4 hours 51Cr release tests of standard.Wherein effector NK cell is tested on known Cw3 responsive to the NK cytolysis or Cw4 positive cell line.The consumption of all target cells is about 5000 cells in each hole of microtitration plate, and the ratio of the effector on target cell illustrates (corresponding 4 effectors of each target cell usually) in the drawings.Have or do not have shown in the condition with the said monoclonal antibody supernatant of 1/2 dilution under carry out cytolysis test.This process basically with unanimity described in document (Moretta et al., 1993).

The generation of new monoclonal antibody

Monoclonal antibody is through usefulness as in document (Moretta et al., 1990; The content of its disclosure is incorporated among this paper with way of reference) described in the Balb C mices in 5 weeks of activatory polyclone or monoclonal NK cell line immunity produce.After different cells merges, thereby monoclonal antibody is owing to they can at first be selected with clone's cross reaction with EB6 and the positive NK cell line of GL183.Positive monoclonal antibody is because the dissolving that can reconstruct be caused by the EB6 positive or the positive NK clone of GL183 of Cw4 or Cw3 positive target cell respectively, thereby further screened.

DF200, a kind of new monoclonal antibody that acts on the shared determining area of KIR2DL people NK receptor

A kind of monoclonal antibody DF200mAb is through finding and can reacting with each member of the KIR family that comprises KIR2DL1, KIR2DL2/3.For painted brightly with the painted NK cell quilt of DF200mAb (KIR2DL1+ and KIR2DL2/3+ cell).(Fig. 1)

The NK clone who expresses one or another kind of (or even two kinds) HLA class I specificity inhibition receptor is as the effector of expressing the allelic anti-target cell of one or more HLA-C.As expect, also be minimum cell lysis activity even if KIR2DL1+NK clone demonstrates the target cell of expressing HLA-Cw4, same, the KIR2DL3+NK clone also demonstrates minimum to the Cw3 positive target cell or does not have activity.Yet when existing DF200mAb when (being used to shelter their KIR2DL receptor), NK clones the HLA-C part that becomes and can not discern them, and on Cw3 or Cw4 target, demonstrates very strong cell lysis activity.

For example, C1R cell line (CW4+EBV cell line, ATCC n ℃ RL 1993) is not killed by KIR2DL1+NK clone (CN5/CN505), but this inhibitory action can be by effective reverse through use DF200 or traditional anti-KIR2DL1mAb.On the other hand, the NK of expressing K IR2DL2/3+KIR2DL1-phenotype clone (CN12) kills C1R effectively, and this causing death do not receive the influence (Fig. 2) of DF200mAb.KIR2DL2-or the positive NK clone of KIR2DL3-that similar result can be used on the positive target of Cw3 obtain.

DF200 mAb/KIR 2DL 1 and the interactional Biacore of DF200 mAb/KIR 2DL3 analyze

Material and method

The production of recombinant protein and purification.KIR2DL 1 produces in the E.coli.cDNA of encoded K IR2 DL and the whole extracellular domains of KIR 2DL3 with the KIR2DL3 recombinant protein, and this KIR2DL and KIR 2DL3 clone 47.11 carriers (Biassoni et al., 1993 by pCDM8 respectively; The content of its disclosure is incorporated among this paper with way of reference) and RSVS (gpt) 183 clones 6 carriers (Wagtman et al., 1995; The content of its disclosure is incorporated among this paper with way of reference), use following primer to obtain:

Justice (primer): 5 '-GGAATTCCAGGAGGAATTTAAAATGCATGAGGGAGTCCACAG-3 ' is arranged

Antisense (primer): 5 '-CCCAAGCTTGGGTTATGTGACAGAAACAAGCAGTGG-3 '.

They in reading frame with sequence clone (Saulquin et al., 2003 in the pML1 expression vector of encoding human elementization signal; The content of its disclosure is incorporated among this paper with way of reference).

In BL21 (DE3) bacterial strain (Invitrogen), accomplish protein expression.The antibacterial of transfection grows into OD in 37 ℃ in the culture fluid that is supplemented with ampicillin (100ug/ml) ₆₀₀=0.6, and induce with 1mM IPTG.

(8M urea) reclaims protein from occlusion body under the degeneration condition.Being folded in again of recombiant protein contained the L-arginine, and (400mM at room temperature realizes through in six steps dialysis (be respectively 4,3,2,1,0.5 and 0M urea), reducing urea concentration gradually Sigma) and among three carboxymethylamino methane (Tris) 20mM of beta-mercaptoethanol (1mM), pH7.8, NaCl 150mM buffer.At 0.5 glutathion that adds through reduction and oxidation during with 0M urea dialysis step (is respectively 5mM and 0.5mM, Sigma).At last, with albumen fully dialysis in three carboxymethylamino methane (Tris) 10mM, pH7.5, NaCl 150mM buffer.With the folded protein again of solubility after concentrating on Superdex 200 exclusion chromatography posts purification (Pharmacia; The AKTA system).

Biacore analyzes.(Biacore) carries out surface plasma resonance mensuration on a Biacore equipment.In all Biacore tests, use the HBS buffer that is supplemented with 0.05% surfactant P20 as running buffer.

Proteopexy.With recombinant KIR2DL1 and KIR2DL3 albumen Covalent Immobilization in the carboxylic group in the glucosan layer of sensor chip CM5 (Sensor Chip CM5) on (Biacore).This sensor chip surface is with EDC/NHS (N-ethyl-N ' (3-dimethyl aminopropyl) carbodiimide hydrochloride and N-maloyl imines, Biacore) activation.Will (the 10mM acetate, the albumen in pH4.5) injects at binding buffer liquid.Using 100mM, pH is the inactivation that 8 ethanolamine (Biocore) carries out remaining active group.

Affinity is measured.In order to carry out kinetic measurement, with the soluble antibody (10 of variable concentrations ^-7To 4 * 10 ^-10M) be applied to this on fixed sample.Continuous flow velocity with 20ul/min is measured.For each circulation, be that 11 NaOH regenerates to the surface of induction chip through 10mM, the pH that injects 5 μ l.

(BIAevaluation 3.1, Biacore) are used for data analysis with BIAlogue kinetics assessment process.

The result

The BIAcore that DF200mAb is incorporated into through fixed K IR 2DL1 and KIR 2DL3 analyzes.

?	KD(10 -9M)
		KIR?2DL1	10.9+/-3.8
KIR?2DL3	2.0+/-1.9

KD: dissociation constant.

With soluble analyte (under the variable concentrations being 40 μ l) with the flow velocity of 20 μ l/min in the HBS buffer be injected into contain respectively KIR 2DL1 and KIR 2DL3 be 500 or 540 catoptries (RU) and 1000 or the glucosan layer of 700RU on.Data represented 6 independently experiments.

Embodiment 2: Mabthera (Rituxan) and anti-KIR monoclonal antibody (anti-KIR mAb) are united use and are strengthened ADCC

Preparation people NK clone.To use negative anti-CD3 immunity magnetic selection method (Miltenyi) to remove blood monocyte bed board under the condition of restricted dilution of T cell; With phytohaemagglutinin A (PHA) (Biochrom KG; Berlin, Germany) activation is with interleukin-22 (IL-2) (Chiron B.V.; Amsterdam Netherlands) cultivates with the feeder cell that shone.Cloning efficiency all equates in all donors, account for bed board the NK cell 1/5 to 1/10 between.Clone's NK cell is the standard of effect in 10: 1 through the B lymph matricyte system to the HLA type Epstein-Barr virus transfection, known to the target cell ratio with effector ⁵¹Cr discharges cell toxicant (property) and screens alloreactivity.Show that >=30% dissolved clone is considered to have alloreactivity.As criterion, the clone refers to or have＜dissolving of 5% demonstration or have＞40%.

Strengthen ADCC with Mabthera (Rituxan) by the positive NK cell clone mediation of KIR2DL1

With the cell lysis activity of standard 4hr 51Cr release measurements determination NK cell, wherein effector NK cell is tested on Cw4 or the positive EBV cell line of Cw3 (CD20 is positive), understands it to the cytolytic sensitivity of NK.All target cells are used with 5000 cells in every hole and the ratio of effector (NK cell clone) on target cell at microtitration plate as shown in Figure 3.In some experiments, (Rituxan Idec) joins in this effector target cell mixture with 5 μ g/ml this therapeutic inosculating antibody CD20 Rituximab.In some experiments, EB6 antibody (anti-KIR2DL1) joins in the effector target cell mixture with 10 μ g/ml.

This experiment demonstrates independent Mabthera (Rituxan) and does not mediate the ADCC through the positive NK clone of KIR2DL1 on the positive target of Cw4 basically.When having anti-KIR2DL1 antibody, the ADCC of KIR2DL1 positive colony has greatly been strengthened.

Embodiment 3: by the enhancing of Kan Pasi (Campath) through the ADCC of the positive NK cell clone mediation of KIR2DL1

With embodiment 2 described similar experiments in; To add that at the NK cell Ah coming organizes monoclonal antibody (alumtuzumab) (Campath from body homology Cw4+PHA blastocyte; Berlex), EB6 antibody (100 μ g/ml) is when existing, or Kan Pasi (Campath) and EB6 are hatched when existing.As shown in Figure 4; The result shows that the existence of EB6 has greatly strengthened the ability that the NK cell is eliminated autologous cell: about 4% target cell dissolving during Kan Pasi (Campaht) individualism, however Kan Pasi (Campath) adds and surpasses 30% cytolysis when EB6 exists.

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All public publications quoted in this description and patent application all are combined in this with their complete forms, are integrated with among this paper through quoting as proof by that kind specific or that point out separately as each independent public publication or patent application.

Although foregoing invention is to have made detailed description to a certain degree for the clear purpose of understanding with chart and the mode of giving an example, those of ordinary skill in the art can do certain change and correction and the spirit or the protection domain that do not deviate from appended claim to it under instruction of the present invention obviously.

Only if do in addition in this article explanation or with the obvious contradiction of content of this paper, in its all possible variation, any combination of above-mentioned key element is all contained in the present invention.

Only if do in addition in this article explanation or with the obvious contradiction of content of this paper, employed term " " is with " being somebody's turn to do " and similarly refer to speech and be understood to include odd number and plural number in describing context of the present invention.

Only if explain in addition in this article; The description of quantitative range only is as the simple expression that falls into each centrifugal pump that relates separately in this scope in this article, and each centrifugal pump is integrated with in this description as that kind of explaining separately in this article.Unless otherwise mentioned; All determined values that provide among this paper are that the representative of corresponding approximation (for example also can be considered to provide corresponding approximate measure value with respect to all exemplary accurate numerical value that specificity factor or measured value provided; In the time of appropriate, can be revised as " pact ").

All methods of describing among this paper can be implemented by any suitable order, only if does in addition in this article to explain or obvious and this paper content contradicts.

Unless otherwise mentioned, use among this paper arbitrarily and all embodiment (example, instance) or exemplary language (for example " as ", " for example ") only be used for describing better the present invention, scope of the present invention is not caused restriction.Only if do clearly statement, not having word can be interpreted as any one key element in this description is that completion is essential to the invention.

Among this paper patent documentation to quote and be combined in this only be for convenience's sake, do not reflect the viewpoint of effectiveness, patentability and/or exploitativeness to these patent documentations.

Of the present invention aspect any one or in the description of embodiment; With respect to a kind of key element or multiple key element use a technical term as " comprising ", " having ", " comprising " or " containing " are for " by ... form "; " basically by ... form " or " comprise that mainly " similar aspect of the present invention or the embodiment of this specific factor or multiple key element provide support; Only if explain at other place or obvious and this paper content (for example contradicts; The compositions that comprises special component described herein also is to be understood that to having described a kind of compositions that is grouped into by this one-tenth, only if explain at other place or obvious and this paper content contradicts).

The present invention includes all changes and the equivalent of the subject matter that allows to greatest extent by applicable law, in the various aspects of this submission or claim, narrated.

P51820HWL.ST25

SEQUENCE?LISTING

< 110>Innate Pharma

Perugia research university

< 120>use the NK cell potentiating compounds to improve the method and composition of efficiency of therapeutic antibodies

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Claims (6)

1. the method for the co-administered chemical compound of selection and therapeutic antibodies, said therapeutic antibodies can partly combine through CD16 and mediation ADCC via the Fc of said therapeutic antibodies, and said method comprises:

I) a kind of antibody of the inhibition receptor that is selected from NKG2A, KIR2DL1, KIR2DL2 and KIR2DL3 of block N K cell is provided;

Ii) have the NK cell and exist/do not existing under the situation of antibody of said block N K cell inhibiting property receptor, with said therapeutic antibodies with can together be hatched by the target cell of said therapeutic antibodies specific recognition;

The antibody of iii) measuring said block N K cell inhibiting property receptor is eliminated the influence of said target cell ability for said NK cell;

The antibody of wherein measuring said block N K cell inhibiting property receptor strengthens said NK cell when eliminating the ability of said target cell, shows that the antibody of said block N K cell inhibiting property receptor is applicable to said method.

2. method according to claim 1, wherein said chemical compound strengthens 30% with the ability that said therapeutic antibodies destroys said target cell.

3. method according to claim 1, wherein said chemical compound strengthens 50% with the ability that said therapeutic antibodies destroys said target cell.

4. according to each described method of claim 1-3, wherein said chemical compound is selected from the one group of material that comprises antibody, antibody fragment, monoclonal antibody, monoclonal antibody fragment, humanized antibody, chimeric antibody and people's antibody.

5. according to each described method of claim 1-4, wherein said target cell is the cell of cancerous cell, viral infection or the cell that constitutes the autoimmune disease basis.

6. according to each described method of claim 1-5, wherein said therapeutic antibodies is Rituximab or Kan Pasi.

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